【作者】 石天尧；

【导师】 招明高；

【作者基本信息】 第四军医大学 ， 药理学， 2013， 博士

【摘要】 目的:阿尔兹海默病(Alzheimer’s disease, AD)是老年期痴呆最常见的类型。认知能力的逐渐下降和淀粉样斑块及神经原纤维缠结的出现是其典型的临床病理特征。最终结果会导致脑功能的异常及神经元凋亡。随着我国人口老龄化的日益严重，AD越来越成为一个严重的社会问题。研究表明AD神经退行性病变过程中，氧化应激是除年龄外另一个重要因素。大量的研究表明DNA、RNA、脂质和蛋白质氧化程度在AD病变过程中不断升高，直至出现轻度认知损伤，这些都说明氧化应激出现在AD的早期阶段，即发生在淀粉样斑块和神经原纤维缠结之前出现。尽管在AD的治疗研究中有很多针对于对抗氧化应激介导损伤的神经保护策略，但是临床结果积极有效的只有很少一部分。氮氧自由基（Nitroxide radicals, NRs）作为一类含有自旋单电子的稳定的自由基化合物。早期被用作自旋示踪剂。近来研究表明NRs还具有其特殊的生物学活性，一些NRs具有抗肿瘤、辐射和缺血再灌注损伤等功能。它与有害自由基反应通过“催化剂量”方式进行，只需很少的剂量即可发挥高效、强效和长效的抗氧化活性。例如，NRs具有拟SOD的作用。它可以通过电子转移的方式参与到细胞内的线粒体的呼吸链式反应，非常快速的清除超氧阴离子自由基O2.-。正是由于NRs以催化方式分解不断产生的有害自由基，反应过程中本身不会被消耗，可以循环再生利用，这也是其它自由基清除剂所无法比拟的。同时因其特有的自旋示踪功能，可通过电子顺磁共振或核磁共振成像实时了解其在组织中的分布及变化反应。正是由于NRs这些独特的性质，我们推测其在AD的发病过程中会发挥很好的抗氧化的能力，起到预防、治疗甚至是诊断AD发生的作用。因此，有望将NRs制备成新型的应用于神经退行性疾病的抗氧化应激损伤的药物。方法：在本实验中，我们采用体外抗氧化模型，Aβ1-42诱导的细胞毒性损伤模型以及APP/PS1双转基因AD鼠模型，来评价NRs的抗氧化能力及对AD的保护作用。体外抗氧化实验中，我们分别采用光泽精诱导的发光体系、CHP诱导的脂质过氧化模型和F2-异前列素免疫试剂盒来评价NRs清除超氧阴离子和抑制脂质过氧化的能力；体外培养原代皮层神经元模型中，在培养到第10天的时候，分别给予10μM的姜黄素、Tempol和L-NNNBP，孵育24小时后，用25μM的寡聚态Aβ1-42处理12小时。换用原培养液继续培养24小时后收集细胞。分别用CCK-8试剂盒检测细胞的存活率；用Tunel试剂盒检测细胞的凋亡；用3-NT的ELISA试剂盒测定组织的硝化应激水平；用阳离子荧光染料-四甲基罗丹明乙酯检测线粒体的膜电位变化；用细胞的免疫荧光观察激活的Caspase-3的变化；最后采用APP/PS-1双转基因AD小鼠来评价NRs的自由基清除能力和对AD的保护作用。实验分为5组：姜黄素治疗组、Tempol治疗组、L-NNNBP治疗组、WT组和TG组。药物用饮用水溶解，终浓度均为1mM，WT组和TG组给予正常饮水。在小鼠6周时给予药物处理，连续给药1个月。用Morris水迷宫实验评价AD小鼠空间学习记忆能力；用刚果红染色方法观察Aβ斑块；用蛋白免疫印迹方法观察磷酸化Tau和GFAP蛋白含量变化；采用免疫组化方法观察星形胶质细胞的激活。结果：体外抗氧化实验中，我们发现L-NNNBP较姜黄素和Tempol能显著减少超氧阴离子和脂质过氧化水平(#P<0.05，##P<0.01)。培养皮层神经元模型中，不同剂量的L-NNNBP对Aβ1-42诱导的细胞毒性都有保护作用(*P<0.05，**P<0.01)，且是浓度依赖的关系。单独给予L-NNNBP，没有对细胞产生毒性作用。而Tempol的三种剂量都没有观察到神经元的保护作用；姜黄素只有在高剂量（10μM）时有细胞保护作用。Tunel法检测细胞凋亡的实验中，相对于姜黄素（42.9%±3.1%）和Tempol（51.1%±1.1%），L-NNNBP高剂量（10μM）处理组，神经元的凋亡显著降低（22.7%±2.6%，*P<0.05,**P<0.01,##P<0.01）。细胞免疫荧光结果说明L-NNNBP的这种抗凋亡的作用是通过降低激活型Caspase-3的表达引起的。Aβ诱导的氧化应激损伤也会造成线粒体功能障碍，影响线粒体正常膜电位，使其去极化。L-NNNBP能显著降低TMRM+的密度，阻止线粒体膜的去极化，保护线粒体的正常功能，且其作用强度显著高于姜黄素和Tempol（#P<0.05）。除了氧化应激，硝化应激也是自由基产生的主要来源。L-NNNBP三个浓度都降低了3-NT的含量（**P<0.01），且抑制率显著高于姜黄素和Tempol（#P<0.05）。Aβ斑块的沉积是AD的主要病理症状之一。在APP/PS1双转基因AD鼠模型中，6个月大的APP/PS-1小鼠给予L-NNNBP(1mM)1个月后，海马和皮层的Aβ斑块的沉积都显著减少，并且效果强于姜黄素和Tempol（##P<0.05）。除了Aβ斑块沉积，AD的另一个病理特征就是Tau蛋白异常过度磷酸化后引起的神经原纤维缠结。APP/PS1小鼠Tau蛋白的Thr205和Ser235两个位点的磷酸化程度显著升高。相对于WT组和其它药物处理组，L-NNNBP显著的降低了Tau蛋白在两个位点的磷酸化程度（**P<0.01，#P<0.05，##P<0.05）。星形胶质细胞的免疫组化和免疫蛋白印迹结果说明其在AD发病的早期就被激活，有可能进一步诱导β-淀粉样蛋白沉积加速和神经纤维缠结形成。而L-NNNBP在发挥抗氧化功效的同时抑制了星形胶质细胞的激活。Morris水迷宫的行为学结果也说明APP/PS1小鼠空间学习和记忆能力受到损伤，在给予L-NNNBP治疗后有显著改善，并且效果强于姜黄素和Tempol（**P<0.01,##P<0.01）。结论：本研究证明，无论是在氧化损伤的体外模型，还是Aβ1-42诱导的细胞毒性损伤模型，或是APP/PS1双转基因AD鼠模型中，新型的手性氮氧自由基L-NNNBP都表现出了令人兴奋的结果。它能清除超氧阴离子、抑制脂质过氧化、减轻Aβ1-42诱导的细胞毒性损伤、抗凋亡、降低氧化或硝化应激损伤、减少Aβ斑块沉积、降低Tau蛋白磷酸化水平、抑制星形胶质细胞激活和改善AD鼠的空间学习和记忆。总之，L-NNNBP很有可能开发成为临床AD治疗的候选药物。更多还原

【Abstract】 Objectives:Alzheimer’s disease （AD）, an age-related neurodegenerative disorder, is the most commonform of dementia. AD is characterized by the deposition of β-amyloid （Aβ） plaques,intracellular neurofibrillary tangles, loss of neurons in the brain, progressive decline ofmemory and cognitive functions, and behavioral and personality changes. In thepathogenesis and progression of AD, aging is the most critical risk factor. Moreover,oxidative stress has an important function in the early stages of AD. Reactive oxygenspecies （ROS）-mediated pathways are involved in AD development. Numerous studieshave reported the presence of elevated DNA, RNA, lipid, and protein oxidation in brainsof subjects with AD and mild cognitive impairment （MCI）, suggesting that oxidative stressis an early event in AD pathogenesis. It illustrate that oxidative stress occurs at early stages, before the appearance of amyloid plaques and neurofibrillary tangles. For mostantioxidant drugs, beneficial effects have been reported in cell cultures, and partially, inanimal models. However, success in human clinical trials is much less frequent.Nitroxide radicals （NRs） are stable free radicals. NRs are utilized as biophysical tools inelectronic spin resonance spectroscopic studies and spin-label oximetry in early days.Recently studies found that NRs has some special biological activities, include radiationprotection, anticancer, against ischemia-reperfusion injury and so on. Unlike otherantioxidants that act in a sacrificial mode, NRs can provide protection in a catalyticmanner. Through the continuous exchange between these forms NRs act asself-replenishing antioxidants that degrade superoxide and peroxide. For example, NRshave Superoxide dismutase （SOD） mimetic action. Similar to endogenous SOD, thenitroxide acts as a catalyst and is not consumed in the process of dismutation of O₂^-toH₂O₂and oxygen. The catalytic rate is higher than SOD. Because its function of spintracer, we can observe its distribution and understand the changes in the body. Therefore,NRs have a better view in anti-oxidative damage as new type antioxidant drugs.Methods:In our study, Lucigenin chemiluminescence models derived from xanthine–xanthineoxidase reaction were used to evaluate the free radical-scavenging activity CHP-inducedlipid peroxidation system to evaluate inhibition of lipid peroxidation of Curcumin, Tempoland L-NNNBP. In primary cortical neuronal cultures, the neurons were rinsed briefly withPBS at the10th day and then pretreated with10μM Curcumin, Tempol and L-NNNBP for24h, respectively, followed by exposure to25μM of Aβ1-42for12h in the same medium.Afterward, cells were washed3times and returned to the original culture medium for24h.Cell viability was determined by cell counting kit-8（CCK-8）. Measurement of apoptoticcells by the TUNEL. Male APP/PS1double-transgenic mice were used in this study toevaluate the protective effect of L-NNNBP. Mice were divided into five groups: Curcumintreatment, Tempol treatment, L-NNNBP treatment, Wild-type and Transgenetic group.Male mice were treated with curcumin, tempol or L-NNNBP （1mM in drinking water）. Treatment was started when the mice were6months old and was continued for1month.After treatment for1month, spatial learning and memory were evaluated by the Morriswater maze test. The mice brain sections were stained with Congo red solution to identifythe Aβ plaques. The culture neurons and hippocampal tissues were normalized viabicinchoninic acid protein assay to generate homogenates.3-Nitrotyrosine （3-NT）measurements were performed in the supernatants.Mitochondrial membrane potential（Δψm） in the culture neurons was detected by the cationic fluorescent probetetramethylrhodamine methyl ester （TMRM）. Western blot analysis was performed to theexpression of phosphorylated Tau and GFAP. Using immunohistochemistry method, wetest the activation of astrocytes.Results:We first examined the ability of L-NNNBP to scavenge the superoxide anion radical.Lucigenin chemiluminescence models derived from xanthine–xanthine oxidase reactionwere also used to evaluate the free radical-scavenging activity of L-NNNBP.L-NNNBPshowed more potent free-radical scavenging activities, which increased thechemiluminescence compared with curcumin and tempol at the same concentrations（#P<0.05，##P<0.01）. In the CHP-induced lipid peroxidation system, L-NNNBP alsoshowed a higher inhibiting rate on lipid peroxidation compared with curcumin and tempol（#P<0.05，##P<0.01）. In cultures, L-NNNBP attenuated Aβ1-42-induced cell death in aconcentration-dependent manner. However, tempol （0.1–10μM） did not exhibit anyneuroprotective activity against Aβ1-42-induced toxicity. Curcumin exhibitedneuroprotection only at a high concentration （10μM）. Treatment with L-NNNBP alonedid not affect the cell viability. TUNEL staining was performed to identify the apoptoticneurons. Pretreatment of L-NNNBP （10μM） significantly reduced apoptotic neurons to22.7%±2.6%, and this anti-apoptotic activity was more significant than that mediated bythe same concentrations of curcumin （42.9%±3.1%） and tempol （51.1%±1.1%）. Cellimmunofluorescence results show that the L-NNNBP have anti-apoptotic effect throughdecreasing the activated Caspase-3. Aβ can cause oxidative stress injury lead tomitochondrial dysfunction, affect the normal mitochondrial membrane potential and make its depolarization. L-NNNBP （0.1–10μM） prevented the depolarization of Δψm caused byAβ1-42treatment, and this action was stronger than those of curcumin and tempol at thesame concentration （#P<0.05）. In addition to oxidative stress, nitrification stress is anothermain source of free radicals generation. LNNNBP attenuated Aβ1-42-induced3-NTincrease. The inhibition of3-NT by curcumin and tempol was weaker compared with thatmediated by L-NNNBP at the same concentration （#P<0.05）.Aβ plaque deposition is one of the main pathological symptom of AD. Treatment withL-NNNBP and tempol markedly reduced Aβ plaque accumulation in the hippocampus andsomatosensory cortex, whereas curcumin reduced Aβ plaque acumulation in thehippocampus only. Besides Aβ plaque deposition, another feature AD pathologicalprogress is neurofibrillary tangles caused by abnormal phosphorylation of Tau. A markeddecrease in Tau phosphorylation at Ser235and Thr205was observed in theL-NNNBP-treated APP/PS1mice compared with the vehicle-treated mice. LNNNBPmarkedly decreased tau phosphorylation compared with curcumin or tempol （**P<0.01，#P<0.05，##P<0.05）.In the brains of AD patients and transgenic AD mouse models, theinfiltration of activated astrocytes are seen in the area of Aβ plaques, which arecharacteristic components of an inflammatory process that develops around an injury inthe brain. Quantitative analysis showed a58.5%±3.2%decrease in GFAP expression inthe L-NNNBP-treated APP/PS1mice compared with that in the control APP/PS1mice（**P<0.01）. In Morris water maze text, the L-NNNBP-treated APP/PS1mice reachedthe platform, which resulted in significantly reduced escape latency across the trialscompared with the control APP/PS1mice, and curcumin-and tempol-treated mice（**P<0.01,##P<0.01）. L-NNNBP markedly improved the learning capability and memoryof APP/PS1mice compared with curcumin or tempol.Conclusions:This study demonstrates that both in oxidative damage vitro model, Aβ1-42-inducedtoxicity model or APP/PS1double transgenic mice model, treatment with the new chiralNRs （L-NNNBP）, shows the exciting results. It can scavenger superoxide anion, inhibitlipid peroxidation, Reduce the cytotoxicity induced by Aβ1-42, anti-apoptosis, reduce oxidation and nitration stress, decrease Aβ plaque deposition, decrease level ofphosphorylated Tau protein, inhibit astrocyte activation and Improve spatial learning andmemory of AD transgenetic mice. In a word, L-NNNBP is likely to become the candidatedrugs for AD clinical therapy.更多还原